This project aims at isolating mutant aminoacyl tRNA synthetases with altered tRNA specificities. The general idea is to mutagenize a synthetase gene that has been cloned on a plasmid. The mutagenized plasmid will then be infected into appropriate hosts and selections will be done to isolate cells that have the expected phenotype. Once such mutants have been isolated and characterized, mutant synthetases will be purified and studied carefully in vitro. In addition, studies are being done with a synthetase mutant that has an altered Km for its amino acid. The question being explored is whether mutations in the amino acid binding site also can affect the tRNA site by, for example, showing some altered tRNA binding specificity. In addition, the mutant synthetase is being used to explore the possibility of interactions of the enzyme with other proteins in the cell.